The present invention relates to a basement membrane composition and method for its preparation and use. More particularly, the present invention is directed to the basement membrane of avian and reptile follicles and the use of same to support the growth and differentiation of eukaryotic cells cultured in vitro.
There has been much research effort directed to finding natural and synthetic materials having the requisite properties for use as cell culture substrates. Cellular morphology and metabolic activity of cultured cells are affected by the composition of the substrate on which they are grown. Presumably cultured cells function best (i.e. proliferate and perform their natural in vivo functions) when cultured on substrates that closely mimic their natural environment.
One biological tissue that can potentially be used to culturing cells is the basement membranes of vertebrate tissues. Basement membranes, also known as basal lamina, are extracellular matrices (ECMs) that compartmentalize tissues and provide important signals for the adhesion, growth, differentiation, migration of cells they support. In addition, basement membranes influence several physiological and pathological processes. Accordingly, complete and intact basement membrane is naturally functions as a substrate for cell proliferation and growth. However, intact basement membranes are not commercially available for research.
The ability of complex substrates to support cell growth in vitro has been previously reported, and matrix products supporting such growth are commercially available. For example, Becton Dickinson currently offers two such products: Human Extracellular Matrix, and MATRIGEL(copyright) Basement Membrane Matrix. Human Extracellular Matrix is a chromatographically partially purified matrix extract derived from human placenta and comprises laminin, collagen IV, and heparin sulfate proteoglycan. (Kleinman, H K et al., U.S. Pat. No. 4,829,000 (1989)) MATRIGEL(copyright) is a soluble basement membrane extract of the Engelbreth-Holm-Swarm (EHS) tumor, gelled to form a reconstituted basement membrane. Both of these matrix products require costly biochemical isolation, purification, and synthesis techniques and thus production costs are high. In addition these matrix products are reconstituted from extracts of basement membranes and thus do not represent the structure of the basement membrane as it exists in the body.
Accordingly, the study of cellular function by culturing cells in vitro is limited by the availability of cell growth substrates that present the appropriate physiological environment for the proliferation and development of the cultured cells.
The present invention is directed to the use of vertebrate basement tissue-derived matrices as substrates for the growth and attachment of a wide variety of cell types. The extracellular matrices for use in accordance with the present invention comprise highly conserved collagens, glycoproteins, proteoglycans, and glycosaminoglycans in their natural configuration and natural concentration. The extracellular matrix is derived from ovarian follicular tissue of a vertebrate species, and in particular ovarian follicular tissue of vertebrate species that lay eggs.
In accordance with one embodiment, a composition comprising intact basement membrane of follicle tissue of a vertebrate is described. The composition comprises basement membrane is substantially free of cells of the source vertebrate wherein the basement membrane is retained in its natural three dimensional shape. Follicle basement membrane (FBM) prepared in accordance with the present invention is use to induce the proliferation and growth of cells. In one embodiment the invention is directed to a composition and method for culturing eukaryotic cells in vitro.